Limits...
Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice.

You-Ten KE, Muise ES, Itié A, Michaliszyn E, Wagner J, Jothy S, Lapp WS, Tremblay ML - J. Exp. Med. (1997)

Bottom Line: However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected.BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency.This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Show MeSH

Related in: MedlinePlus

Generation of TC-PTP–deficient mutant mice by gene targeting. (A) Targeting construct of TC-PTP. Diagram of wt TC-PTP  genomic locus encompassing four exons (nc 227–771, aa 54–235, in TC-PTP cDNA; reference 14). Probe A hybridizes to a 5.2-kb fragment generated by NdeI digestion. The targeting construct using a 5.5-kb genomic  sequence with the neo resistance gene eliminates ∼9 kb of genomic sequence including 1.5 exons. The resulting targeted locus after a correct  homologous recombination event is shown. In the targeted allele, probe  A hybridizes to a 7.6-kb fragment generated by NdeI digestion. H3, HindIII. The thick line in the targeting construct diagram represents pBluescriptTM IIKS(+) backbone sequences. (B) Southern blot analysis of representative ES cell clones after electroporation. For each clone, DNA was  extracted from one well of a 96-well plate and digested NdeI (+/+, wt;  +/−, heterozygously targeted clone [arrow]). Hybridization was with  probe A. Targeted allele and normal allele correspond to the 7.6-kb and  5.2-kb fragments, respectively. (C) Southern blot analysis of mouse tail  DNA from progeny of heterozygous mouse matings. After DNA extraction, 50 μg of tail DNA were digested with NdeI (+/+, wt; +/−, heterozygotes; −/−, homozygotes). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb  fragments, respectively. (D) Northern blot analysis of total RNA extracted from spleen of wt (+/+) and homozygous (−/−) mice. Wt TC-PTP message was detected as a 1.5-kb transcript using TC-PTP cDNA as  a probe. The same blot was stripped and reprobed with a glyceraldehyde  3-phosphate dehydrogenase probe to assess the level of loading. (E) Western blot analysis of total cellular protein extracted from spleen of wt (+/ +) and homozygous (−/−) mice using the anti TC-PTP monoclonal antibody 3E2. TC-PTP migrates at 45 kD. Other bands represent immunogloblin chain subunits that are recognized by the secondary goat anti– mouse horseradish peroxidase conjugated antibody. (F) Table of progeny  survival in littermates from the crossing of heterozygous mice for the TC-PTP disruption.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199020&req=5

Figure 1: Generation of TC-PTP–deficient mutant mice by gene targeting. (A) Targeting construct of TC-PTP. Diagram of wt TC-PTP genomic locus encompassing four exons (nc 227–771, aa 54–235, in TC-PTP cDNA; reference 14). Probe A hybridizes to a 5.2-kb fragment generated by NdeI digestion. The targeting construct using a 5.5-kb genomic sequence with the neo resistance gene eliminates ∼9 kb of genomic sequence including 1.5 exons. The resulting targeted locus after a correct homologous recombination event is shown. In the targeted allele, probe A hybridizes to a 7.6-kb fragment generated by NdeI digestion. H3, HindIII. The thick line in the targeting construct diagram represents pBluescriptTM IIKS(+) backbone sequences. (B) Southern blot analysis of representative ES cell clones after electroporation. For each clone, DNA was extracted from one well of a 96-well plate and digested NdeI (+/+, wt; +/−, heterozygously targeted clone [arrow]). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (C) Southern blot analysis of mouse tail DNA from progeny of heterozygous mouse matings. After DNA extraction, 50 μg of tail DNA were digested with NdeI (+/+, wt; +/−, heterozygotes; −/−, homozygotes). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (D) Northern blot analysis of total RNA extracted from spleen of wt (+/+) and homozygous (−/−) mice. Wt TC-PTP message was detected as a 1.5-kb transcript using TC-PTP cDNA as a probe. The same blot was stripped and reprobed with a glyceraldehyde 3-phosphate dehydrogenase probe to assess the level of loading. (E) Western blot analysis of total cellular protein extracted from spleen of wt (+/ +) and homozygous (−/−) mice using the anti TC-PTP monoclonal antibody 3E2. TC-PTP migrates at 45 kD. Other bands represent immunogloblin chain subunits that are recognized by the secondary goat anti– mouse horseradish peroxidase conjugated antibody. (F) Table of progeny survival in littermates from the crossing of heterozygous mice for the TC-PTP disruption.

Mentions: The intron/exon boundaries of genomic clones were determined by Southern mapping and DNA sequencing. Dideoxy sequencing reactions were performed using α-[35S]dATP (New England Nuclear) and Sequenase™ version 2.0 (Amersham Corp., Arlington Heights, IL) as stated in the manufacturer's instructions. The TC-PTP targeting construct as shown in Fig. 1 A was generated by subcloning, into pBluescriptTM II KS(+) (Stratagene Corp.), the neomycin (neo) resistance gene cassette flanked by TC-PTP genomic sequences. pMC1neoPolyA (Stratagene Corp.) contains a 1-kb cassette with the neomycin resistance gene under the control of the Herpes simplex thymidine kinase gene promoter. The plasmid was digested with XhoI, the overhangs were blunt ended with deoxyribonucleotides (Pharmacia) and the Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA), and then digested with HindIII. The 1.1-kb fragment was gel purified and ligated using T4 DNA ligase (New England Biolabs) to pBluescriptTM II KS(+) that had been digested with PstI, rendered blunt ended as described above, and digested with HindIII, generating the plasmid BSNEO.


Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice.

You-Ten KE, Muise ES, Itié A, Michaliszyn E, Wagner J, Jothy S, Lapp WS, Tremblay ML - J. Exp. Med. (1997)

Generation of TC-PTP–deficient mutant mice by gene targeting. (A) Targeting construct of TC-PTP. Diagram of wt TC-PTP  genomic locus encompassing four exons (nc 227–771, aa 54–235, in TC-PTP cDNA; reference 14). Probe A hybridizes to a 5.2-kb fragment generated by NdeI digestion. The targeting construct using a 5.5-kb genomic  sequence with the neo resistance gene eliminates ∼9 kb of genomic sequence including 1.5 exons. The resulting targeted locus after a correct  homologous recombination event is shown. In the targeted allele, probe  A hybridizes to a 7.6-kb fragment generated by NdeI digestion. H3, HindIII. The thick line in the targeting construct diagram represents pBluescriptTM IIKS(+) backbone sequences. (B) Southern blot analysis of representative ES cell clones after electroporation. For each clone, DNA was  extracted from one well of a 96-well plate and digested NdeI (+/+, wt;  +/−, heterozygously targeted clone [arrow]). Hybridization was with  probe A. Targeted allele and normal allele correspond to the 7.6-kb and  5.2-kb fragments, respectively. (C) Southern blot analysis of mouse tail  DNA from progeny of heterozygous mouse matings. After DNA extraction, 50 μg of tail DNA were digested with NdeI (+/+, wt; +/−, heterozygotes; −/−, homozygotes). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb  fragments, respectively. (D) Northern blot analysis of total RNA extracted from spleen of wt (+/+) and homozygous (−/−) mice. Wt TC-PTP message was detected as a 1.5-kb transcript using TC-PTP cDNA as  a probe. The same blot was stripped and reprobed with a glyceraldehyde  3-phosphate dehydrogenase probe to assess the level of loading. (E) Western blot analysis of total cellular protein extracted from spleen of wt (+/ +) and homozygous (−/−) mice using the anti TC-PTP monoclonal antibody 3E2. TC-PTP migrates at 45 kD. Other bands represent immunogloblin chain subunits that are recognized by the secondary goat anti– mouse horseradish peroxidase conjugated antibody. (F) Table of progeny  survival in littermates from the crossing of heterozygous mice for the TC-PTP disruption.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199020&req=5

Figure 1: Generation of TC-PTP–deficient mutant mice by gene targeting. (A) Targeting construct of TC-PTP. Diagram of wt TC-PTP genomic locus encompassing four exons (nc 227–771, aa 54–235, in TC-PTP cDNA; reference 14). Probe A hybridizes to a 5.2-kb fragment generated by NdeI digestion. The targeting construct using a 5.5-kb genomic sequence with the neo resistance gene eliminates ∼9 kb of genomic sequence including 1.5 exons. The resulting targeted locus after a correct homologous recombination event is shown. In the targeted allele, probe A hybridizes to a 7.6-kb fragment generated by NdeI digestion. H3, HindIII. The thick line in the targeting construct diagram represents pBluescriptTM IIKS(+) backbone sequences. (B) Southern blot analysis of representative ES cell clones after electroporation. For each clone, DNA was extracted from one well of a 96-well plate and digested NdeI (+/+, wt; +/−, heterozygously targeted clone [arrow]). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (C) Southern blot analysis of mouse tail DNA from progeny of heterozygous mouse matings. After DNA extraction, 50 μg of tail DNA were digested with NdeI (+/+, wt; +/−, heterozygotes; −/−, homozygotes). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (D) Northern blot analysis of total RNA extracted from spleen of wt (+/+) and homozygous (−/−) mice. Wt TC-PTP message was detected as a 1.5-kb transcript using TC-PTP cDNA as a probe. The same blot was stripped and reprobed with a glyceraldehyde 3-phosphate dehydrogenase probe to assess the level of loading. (E) Western blot analysis of total cellular protein extracted from spleen of wt (+/ +) and homozygous (−/−) mice using the anti TC-PTP monoclonal antibody 3E2. TC-PTP migrates at 45 kD. Other bands represent immunogloblin chain subunits that are recognized by the secondary goat anti– mouse horseradish peroxidase conjugated antibody. (F) Table of progeny survival in littermates from the crossing of heterozygous mice for the TC-PTP disruption.
Mentions: The intron/exon boundaries of genomic clones were determined by Southern mapping and DNA sequencing. Dideoxy sequencing reactions were performed using α-[35S]dATP (New England Nuclear) and Sequenase™ version 2.0 (Amersham Corp., Arlington Heights, IL) as stated in the manufacturer's instructions. The TC-PTP targeting construct as shown in Fig. 1 A was generated by subcloning, into pBluescriptTM II KS(+) (Stratagene Corp.), the neomycin (neo) resistance gene cassette flanked by TC-PTP genomic sequences. pMC1neoPolyA (Stratagene Corp.) contains a 1-kb cassette with the neomycin resistance gene under the control of the Herpes simplex thymidine kinase gene promoter. The plasmid was digested with XhoI, the overhangs were blunt ended with deoxyribonucleotides (Pharmacia) and the Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA), and then digested with HindIII. The 1.1-kb fragment was gel purified and ligated using T4 DNA ligase (New England Biolabs) to pBluescriptTM II KS(+) that had been digested with PstI, rendered blunt ended as described above, and digested with HindIII, generating the plasmid BSNEO.

Bottom Line: However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected.BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency.This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Show MeSH
Related in: MedlinePlus